A Study on the Interaction between Transcriptional Factor and Protein of Tachi2 Chitinase Gene in Trichoderma asperellum

doi:10.13560/j.cnki.biotech.bull.1985.2024-0992

Abstract

Abstract:

Objective The chitinase Tachi2, produced by the biocontrol fungus Trichoderma asperellum TD3104, is recognized as playing a significant role in the process of plant disease control. The transcription factor 47 is known to act on the specific response to chitin-induced Tachi2 gene promoter cis-acting elements. The clarification of the interaction between transcription factor 47 and a novel regulatory protein H63 is sought, providing a scientific basis for elucidating the transcriptional expression mechanism of chitin induced regulatory genes. Method The yeast two-hybrid system was employed to identify the candidate interacting protein H63 of transcription factor 47 within the chitinase gene Tachi2 of T. asperellumin vivo. Prokaryotic induction expression of H63 protein and transcription factor 47 gene was performed using an Escherichia coli expression system. The fusion protein was purified using affinity chromatography technology, and protein interactions were detected in vitro using GST pull-down experiments. Further verification of the interaction relationship was conducted through Agrobacterium mediated subcellular localization technique and BiFC experiment of onion epidermal cells. Result It was observed that the H63 protein interacts with transcription factor 47 within yeast cells. The recombinant H63 protein and transcription factor 47 expressed in prokaryotic cells have sizes of 36 kD and 18 kD, respectively, and there is an interaction between the two in vitro. A bimolecular fluorescent complementary vector was successfully constructed, the interaction between H63 protein and transcription factor 47 in onion epidermal cells was confirmed, and the interaction occurred in the nucleus. Conclusion This confirms the interaction between H63 protein and transcription factor 47 both inside and outside the cell, laying a theoretical foundation for analyzing the expression regulation of fungal chitinase genes and the development and utilization of chitinase in agriculture and biomedicine.

Key words: Trichoderma asperellum, chitinase, transcription factor, gene expression, interacting proteins, yeast two hybrid, GST pull-down, BiFC

QU Shan, ZHAO Yue, LI Ya-hua, ZHENG Gui-ling, XIAN Hong-quan. A Study on the Interaction between Transcriptional Factor and Protein of Tachi2 Chitinase Gene in Trichoderma asperellum[J]. Biotechnology Bulletin, 2025, 41(5): 310-319.

Figures/Tables 11

Table 1 Primers used in the experiment

引物名称 Primer name	序列 Sequence （5′-3′）	限制性内切酶 Restriction enzyme	用途 Usage
47S	CCGGAATTCATGCCGGATGCGGTTACAGAG	EcoR Ⅰ	点对点验证
47A	CGGGATCCTCAAGGCCCACACACCTCCGA	BamH Ⅰ	点对点验证
H63S	TCCCCCCGGGATGGGGGTCCGGCGGC	Xma I	点对点验证
H63A	CGGGATCCTACGGATGCGAAAAGTTGGT	BamH Ⅰ	点对点验证
5′AD	CTATTCGATGATGAAGATACCCCACCAAACCC	-	点对点验证
3′AD	AGTGAACTTGGGGGGTTTTTCAGTATCTACGAT	-	点对点验证
H63S-B/N	CGGGATCCATGGGGTCCGGCGGC	BamH Ⅰ	GST pull-down
H63A-B/N	ATTTGCGGCCGCCTACGGATGCGAAAAGTTGGT	Not I	GST pull-down
CE63S	CGGGATCCATGGGGGTCCGGCGGC	BamH I	BiFC
CE63A	GGGGTACCATCGAAAATATGACCGCACT	Kpn I	BiFC
NE47S	CGGGATCCATGCCGGATGCGGTTACAGAG	BamH I	BiFC
NE47A	GGGGTACCAGGCCCACACACCTCCGA	Kpn I	BiFC

Table 2 GST pull-down experimental design

组别 Group	蛋白 Protein
实验组1	GST-H63蛋白+His-47蛋白
实验组2	GST标签蛋白+His-47蛋白
实验组3	GST标签蛋白
对照组1	纯化的His-47蛋白
对照组2	纯化的GST-H63蛋白

Fig. 1 pGBKT7-47 vector constructionA: RNA of Trichoderma asperellum; B: PGBKT7-47 double enzyme digestion verification (pGBKT7-47 double enzyme-digested product; M: 5000 bp DNA marker)

Fig. 2 pGADT7-47 vector self-activation and toxicity testA: Y2H Gold yeast transformant of pGBKT7-47 and pGADT7; B: Y2H Gold yeast transformant of pGADT7-T and pGBKT7-lam; C: Y2H Gold yeast transformant of pGADT7-T and pGBKT7-53

Fig. 3 Construction of interacting pGADT7-H63 vectorA: H63 gene PCR product; B: H63 bacterial liquid PCR product validation; C: PGADT7-63 double enzyme digestion product; M: DL5000 bp DNA marker

Fig. 4 Verification of the interaction of pGBKT7-47 with pGADT7-H63 through yeast two-hybridA: Y2H Gold yeast transformant of pGBKT7-47 and pGADT7-H63; B: Y2H Gold yeast transformant of pGADT7-T and pGBKT7-53

Fig. 5 Construction of pGEX4T-H63 vectorA: H63 gene PCR product; B: pGEX4T-H63 double enzyme digestion product; M: DL5000 bp DNA marker

Fig. 6 SDS-PAGE electrophoresis map of recombinant proteinA: Purified 47 protein; B: purified pGEX4T-1-H63 protein; C: purified pGEX4T-1 protein; M: standard protein marker

Fig. 7 Protein interaction validation of GST-H63 and His-47 in vitro1: GST-H63 protein and His-47 protein; 2: His-47 protein and GST protein; 3: GST; 4: His-47 protein; 5: GST-H63 protein; M: standard protein marker

Fig. 8 Construction of BiFC experimental carrierA: H63 gene PCR product; B: PCR product of transcription factor gene 47; C: PCR validation of bacterial liquid (1: PCR product of DH5 α/B-T1-47 bacterial liquid; 2: PCR product of DH5 α/B-T1-H63 bacterial liquid); D: double enzyme digestion validation (1: NE-47 double enzyme cleavage product; 2: CE-63 double enzyme cleavage product); M: DL5000bp DNA marker

Fig. 9 BiFC experiment validating protein interaction

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